Research Article In Vitro Efficacy of Medicinal Plant Material on the

Research Article
The Journal of Advances in Parasitology
In Vitro Efficacy of Medicinal Plant Material on the Inhibition of
Development of Egg of Ascaridia galli
Md. Shakhayet Hossain1, Anita Rani Dey2*, Nurjahan Begum2, Md. Abdul Alim2
Agroni bank Ltd, Bandar branch, Chittagong; 2Department of Parasitology, Bangladesh Agricultural University,
Mymensingh-2202, Bangladesh.
1
Abstract | To determine the in vitro efficacy of indigenous plants on the inhibition of development of eggs of Ascaridia
galli, a study was carried out in the laboratory of Department of Parasitology from January to May, 2012. Leaves of five
plants namely pineapple (Ananas comosus), turmeric (Curcuma domestica), akanda (Calotropis gigantea), garlic (Allium
sativum) and custard apple (Annona reticulata) were selected. Two different types of preparation such as fresh juice and
dust material were used. Fresh leaves juice at 5%, 10% and 20% concentration and dust of leaves at 5%, 10% and 20%
concentration were used for screening. Among the selected plants in all three concentrations of fresh juice of leaves,
pineapple was the highest effective plant (86.9%) at 20% concentration followed by pineapple at 10% concentration
(84.0%), garlic at 5% concentration (81.8%), turmeric at 20% concentration (78.9%), pineapple at 5% concentration
(78.9%), turmeric at 10% concentration (73.9%), garlic at 10% concentration (73.0%). Among the plants in all concentrations of dust of leaves, pineapple at 20% concentration (76.9%) was observed as the best plant followed by akanda
at 5% concentration (75.0%) and turmeric at 20% concentration (73.3%). The present study suggests that pineapple,
turmeric and garlic leaves are effective and can be used against the development of eggs of A. galli. So, further studies
are required to determine the side effects of these plants and determine the recommended doses in poultry.
Keywords | In vitro, Efficacy, Medicinal plant, Ascaridia galli
Editor | Muhammad Imran Rashid, Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
Received | December 08, 2014; Revised | March 09, 2015; Accepted | March 10, 2015; Published | April 07, 2015
*Correspondence | Anita Rani Dey, Bangladesh Agricultural University, Mymensingh, Bangladesh; Email: [email protected]
Citation | Hossain MS, Dey AR, Alim MA, Begum N (2015). In vitro efficacy of medicinal plant material on the inhibition of development of egg of Ascaridia
galli. J. Adv. Parasitol. 2(1): 5-10.
DOI | http://dx.doi.org/10.14737/journal.jap/2015/2.1.5.10
ISSN | 2311–4096
Copyright © 2015 Hossain et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
INTRODUCTION
ufactured drugs have become evident in the world, such as
drug resistance, food residues and environmental pollution.
scaridia galli is one of the most common parasitic A general stagnation in the development of conventional
roundworms of poultry that occurs in chickens and medicine has led to an increased need for research into alturkey (Permin, 1997; Soulsby, 1982; Anderson, 1992). ternative therapeutic agents for the treatment and control
Embryonated eggs of A. galli are very hardy and under of helminth infections. Medicinal plants have been used
laboratory conditions may live for two years in ordinary to combat parasitism, and in many parts of the world are
conditions. However, few probably live more than one still used for this purpose (Athanasiadou et al., 2007). In
year (Matter and Oester, 1989). Disinfectants and other ethno-veterinary medicine, which draws inspiration from
cleaning agents do not kill eggs under farm conditions and traditional practice, there seems to be a range of plant/s
chickens become infected by eating infective eggs contain- or plant extract suitable for treating almost every parasitic
ing L3 (Permin, 1997). Chemical anthelmintics have long disease of livestock and poultry.
been considered the only effective way of controlling this
parasitic infection. Available drugs remove only the adult It is estimated that more than 20,000 species of plants
parasite and also some serious disadvantages of using man- are used medicinally throughout the world for controlling
A
January 2015 | Volume 2 | Issue 1 | Page 5
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diverse disease ( Jeyathilakan et al., 2012). Pineapple, tur- Collection and Maintenance of Eggs of A. galli
meric, akanda, garlic and custard apple are the medicinal
plants available in Bangladesh those have anthelmintic activity (Anthony et al., 2005; Kiuchi et al., 1993; Patra et
al., 2010). The active compound of garlic is allicin which is
an organosulfur compound (Miron et al., 2000). The pharmacological activity of turmeric has nematocidal (Kiuchi
et al., 1993). Treatment with allicin from garlic is effective
against A. galli in chicken (Velkers et al., 2011). In vitro and
in vivo screening of plant materials as anthelmintic against
adult A. galli has been done throughout the world with the
aim of controlling A. galli in poultry. But eggs generally
have a long survival rate in the environment, and thus a
high infection potential. So, the present study will have a
great importance in controlling A. galli in chickens, as this
work will be a stand point to control A. galli by inhibiting
the development of eggs using available indigenous plants
and thus reducing the infection. Therefore, this research
plan was designed with a view to evaluate the efficacy of
some plant materials on the inhibition of development of
eggs of A. galli eggs and also to compare the efficacy of the
fresh juice and dust of leaves of candidate plants against
development of A. galli eggs.
MATERIALS AND METHODS
The study was conducted from January May, 2012 in the
laboratory of the Department of Parasitology, Bangladesh
Agricultural University (BAU), Mymensingh.
Preparation of Plant Materials for Experiment
Selection of Plants Used in this Experiment
Five plants namely pineapple (A. comosus), turmeric (C. domestica), akanda (C. gigantea), garlic (A. sativum) and custard apple (A. reticulata) were selected on the basis of their
ethnomedical uses for screening.
Processing of Leaves
After collection, leaves were separated from the plants and
washed thoroughly in running tap water. The dust and
juice of leaves used in this study were processed. For the
preparation of fresh juice, the fresh leaves were cut into
small pieces and water was added at 1:1 ratio in a kitchen
blender. Then juice were made by blending the leaves for
2-3 minutes and stored in a refrigerator at 4°C to maintain
the quality of active ingredients of juice. For the preparation of dust, the leaves were dried in the shade at room
temperature and then they were dried in the oven at 5560°C. The dried leaves were cut into small pieces and pulverized with a blender. A 25mm mesh diameter sieve was
used to obtain the fine dust, after then dust was preserved
in airtight plastic container until they were directly used in
litter materials for screening and preparation of aqueous.
January 2015 | Volume 2 | Issue 1 | Page 6
Collection of Eggs of A. galli
At first, intestines were collected from indigenous chickens slaughtered in the nearby market at BAU campus and
brought to the laboratory. Then, adult A. galli were collected
from small intestine following a standard method (Fowler,
1990). Female parasites were identified under microscope
and separated from males. Eggs were recovered by grinding the female parasites with pestle and mortar by adding
5 ml PBS. These fresh eggs were used for the experiment.
Treatment of Egg-PBS Suspension with Plant
Materials
Treatment of eggs with fresh leaves juice: Petri dishes
were used for the treatment which were properly washed,
dried and then labeled. PBS was used as media for this
trial and 10 ml of total volume were made for each trial as
in Table 1. Upper meniscus of total 10 ml volume of suspension in all petridishes was marked by permanent ink.
Fresh leaves juices were used as 5%, 10% and 20% and for
control, one petridish with egg-PBS suspension retained
without treatment. The petridishes were kept at room temperature for 20 days. Petri dishes were placed at a large
tray and moist cotton was used under the petri dishes to
prevent desiccation. All petri dishes were covered in same
manner; about half of the petri dishes were kept open to
allow aeration for development of eggs. Continuous monitoring of petri dishes had been done and the upper meniscus of fluid was maintained by adding PBS if necessary.
Table 1: Making of fresh leaves juice concentrations for
trial
ConcentraVolume (in ml)
tion (in %) Fresh leaves PBS Egg-PBS
Juice
suspension
Control (0)
5
10
20
0
0.5
1
2
9
8.5
8
7
1
1
1
1
Total
10
10
10
10
Treatment of litter with dust: Dust of each plant leaves
were used at 10% and 20% concentrations. For making different preparations, 8g, 9g and 9.5g, 5% of litter
were mixed with 2g, 1g and 0.5g of dust for 20%, 10%
and 0.5% concentration respectively. For each trial, total 10g of litter-dust mixture were kept in the medium
sized petri dishes and 1 ml of egg-PBS suspensions were
sprayed on each petri dish. For the control, only 10g of
litter were sprayed with 1 ml of egg-PBS suspension. All
petri dishes were kept at room temperature for 20 days
and after then water was added with the mixture and
sieved to remove litter. The filtrate was allowed to stand
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for 30 minutes for sedimentation of eggs. The supernatant
was poured off and the sediment was washed for several
times to make it clear. Finally the filtrate was centrifuged
at 1500 rpm for five minutes and the sediment was taken on a clear slide to examine the eggs under microscope.
The Journal of Advances in Parasitology
cy was found in pineapple (86.9%) followed by turmeric
(78.9%), custard apple (72.0%), akanda (66.6%) and garlic
(65.0%) (Table 2).
Overall Performance of Fresh Leaves Juice
Among the selected plants and in all three concentrations
Examination of eggs for development of larvae: Fresh of fresh juice of leaves, pineapple was the highest efficacious
juice and dust treated eggs were examined at 10th day, plant against the development of A. galli eggs. Papaya was
15th day and 20th day for the development of larvae with- the second highest followed by akanda and garlic, whereas
in the eggs. Developed eggs were identified with the custard apple was the least efficacious. The efficacy of fresh
presence of larvae within egg and the movement of lar- juice of leaves of plants can be presented chronologically
vae. Both fresh/undeveloped and treated eggs were iden- as: pineapple at 20% concentration (86.9%) > pineapple at
tified under microscope with high power objectives. 10% concentration (84.0%) > garlic at 5% concentration
(81.8%) > turmeric at 20% concentration (78.9%) > pineDetermination of efficacy of plants: In vitro screening of apple at 5% concentration (78.9%) > turmeric at 10% confresh leaves juice and dust of leaves of selected plants on centration (73.9%) >garlic at 10% concentration (73.0%).
the inhibition of development of A. galli eggs were done
for their efficacy; and the plant/preparations were consid- In vitro Screening of Dust of Leaves
ered as effective having at least 70% efficacy.
Efficacy of Dust at 5% Concentration
At 5% concentration of dust of leaves, the highest efficaStatistical Analysis
In vitro effects of different preparations of plant leaves cy was observed in akanda (75.0%), followed by turmeric
were statistically analyzed with ANOVA technique to (73.3%), pineapple (70.5%), garlic (64.2%) and custard apobtain the level of significance using MSTAT-C package ple (57.1%) (Table 3).
programme developed by Russell (1986). The mean differEfficacy of Dust at 10% Concentration
ences were compared by /Duncan’s New Multiple Range
At 10% concentration of dust of leaves, the highest efficaTest (DMRT) (Gomez and Gomez, 1984).
cy was observed in pineapple and turmeric (71.4%), followed by akanda (60.0%), custard apple (53.8%) and garlic
RESULTS
(50.0%) (Table 3).
Among the five selected plant, pineapple was the highest
Efficacy of Dust at 20% Concentration
efficacious plant against the development of A. galli eggs
In case of dust preparation of leaves at 20% concentration,
at 20% concentration (86.9%) of fresh juice of leaves folthe highest efficacy was found in the pineapple (76.9%),
lowed by pineapple at 10% concentration (84.0%), garlic
followed by turmeric (73.3%), akanda 69.2%), garlic
at 5% concentration (81.8%), turmeric at 20% concentra(66.6%) and custard apple (53.3%) (Table 3).
tion (78.9%) (Table 2). From the present study, it is found
that fresh leaves juice was more efficacious than the dust Overall Efficacy of Dust of Leaves
of leaves.
Among the plants, in all concentrations of dust of leaves,
Pineapple was observed as the best plant followed by
akanda, turmeric, garlic and custard apple. Chronological
Efficacy of Fresh Leaves Juice at 5% Concentrations distribution of efficacy of dust of plants leaves at different
During in vitro screening of fresh leaves juice of five select- concentrations: pineapple at 20% concentration (76.9%)
ed plants at 5% concentration, the highest efficacy in terms > akanda at 5% concentration (75.0%) > turmeric at 20%
of inhibition of development of growth of larvae was found concentration (73.3%). In case of control group, less than
in garlic (81.8%) followed by pineapple (78.9%), turmeric 22% efficacy was found in all concentration of juice and
(70.0%), akanda (69.2%) and custard apple (58.3%) (Table 2). dust preparation.
In vitro Screening of Fresh Juice of Leaves
Efficacy of Fresh Leaves Juice at 10% Concentration
In case of investigation of efficacy of fresh leaves juice at
10% concentration, pineapple was found as most efficacious plant (84.0%) followed by turmeric (73.9%), akanda
(73.0%), custard apple (71.4%) and garlic (63.1%) (Table 2).
Efficacy of Fresh Leaves Juice at 20% Concentration
At 20% concentration of fresh leaves juice, highest effica-
January 2015 | Volume 2 | Issue 1 | Page 7
DISCUSSION
Pineapple (A. comosus) is an important medicinal plant with
diverse pharmacological spectrum. The active ingredient of
A. comosus is bromelian. The effect of bromelian (proteolytic
enzyme) is to digest parasites (Lechat et al., 1978). In this
experiment, highest efficacy of pineapple leaves was found
in 20% juice (86.9%), followed by 10% juice (84.0%). These
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15th
14
7
3
6
5
4
20th
15
10
4
8
6
4
Observation at (days)
12
4
1
2
4
2
10th
No. of developed
eggs
5% concentration
Total
no. of
egg
20
19
26
22
24
17
No. of
undeveloped
eggs
Effects
against
development of
eggs
11.7
58.3
81.8
69.2
70.0
78.9
2
14
18
18
14
15
23
17
21
19
26
25
Total
no. of
egg
13
4
5
3
2
2
10th
15
5
7
6
4
4
15th
15
6
7
7
6
4
20th
Observation at (days)
No. of developed
eggs
10% concentration
10th
8
4
6
4
2
2
15th
19
6
7
6
3
3
20th
23
7
7
6
4
3
Effects Total No. of developed
against no. of eggs
develop- egg
ment of
eggs
Observation at (days)
19
25
25
20
18
23
No. of
undeveloped
eggs
11.7
71.4
63.1
73.0
73.9
84.0
2
15
12
19
17
21
20% concentration
Table 2: Efficacy of fresh leaves juice of five selected plants at 5%, 10% and 20% concentration against development of A. galli eggs
Name of
plants
Turmeric
Pineapple
Akanda
Garlic
Custard apple
Control
No. of
undeveloped
eggs
Effects
against
development of
eggs
8.0
72.0
65.0
66.6
78.9
86.9
15
20
12
13
18
2
Level of significance
**
**
**
In a column figures with same letter or without letter do not differ significantly whereas figures with dissimilar letter differ significantly (as per DMRT); ** = Significant at 1% level
of probability
15th
5
20th
5
Observation at (days)
3
10th
No. of developed
eggs
5% concentration
Total
no. of
egg
17
4
12
11
No. of
undeveloped
eggs
2
3
10th
3
4
15th
4
4
20th
Observation at (days)
10% concentration
14
14
Effects Total
on
no. of No. of developed eggs
develop- egg
ment of
eggs
70.5
73.3
10
10
No. of
undeveloped
eggs
71.4
71.4
Effects
on development
of eggs
13
15
10th
3
3
15th
3
4
20th
Observation at (days)
2
2
10
11
73.3
76.9
Total No. of developed eggs No. of
Effects on
no. of
undedevelopegg
veloped ment of
eggs
eggs
20% concentration
Table 3: Efficacy of dust of leaves of five selected plants against development at 5%, 10% and 20% concentration of A. galli eggs
Name of
plants
Pineapple
3
15
2
Turmeric
66.6
53.3
69.2
8
21.4
8
9
4
3
7
4
2
11
6
4
1
9
3
3
12
8
15
13
50.0
14
53.8
60.0
8
16.6
7
9
8
2
6
6
8
10
5
5
6
9
4
2
16
7
13
15
64.2
12
57.1
75.0
9
15.3
8
12
5
2
6
4
4
11
6
3
3
9
4
1
14
8
16
Garlic
13
Custard apple 14
Akanda
Control
Level of significance
**
**
**
In a column figures with same letter or without letter do not differ significantly whereas figures with dissimilar letter differ significantly (as per DMRT); ** = Significant at 1% level
of probability
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finding could be compared with the study of Rahman
(2002) who recorded the findings against gastro intestinal nematodes where juice of pineapple showed the best
(86.00%) efficacy. Patra et al. (2010) reported that the anthelmintic activity of pineapple is 85.0% after 20 days of
treatment.
Curcumin is the main active constituent of turmeric which
may cause death of A. galli during in vitro study (Bazh and
El-Bahy, 2013). Number of researchers reported the anthelmintic activity of turmeric (C. domestica) against adult
A. galli (Ahktar and Riffat, 1985; Ali, 2006) and other helminths (Rahman, 2002; Hordegen et al., 2003; Sharma et
al., 2003; Mishra et al., 2004; Githiori et al., 2004; Chandrawathani et al., 2006; Szewczuk et al., 2006). In this experiment, highest efficacy of turmeric leaves was found in
20% juice (78.9%), followed by 10% juice (73.9%) and dust
at 20% concentration (73.3%). These findings indicated
that turmeric leaves have better efficacy on the inhibition
of development of A. galli eggs in juice preparation than
dust. Rahman (2002) recorded the highest efficacy (100%)
of turmeric leaves in alcoholic extract whereas aqueous extract have the lower efficacy (92.0%) against gastrointestinal nematodes in goats.
The active ingredient of garlic (A. sativum) is allicin. Allicin is readily permeable through phospholipid membranes
(Oommen et al., 2004) and affects helminthes by altering the body surface of helminthes (Shalaby and Farag,
2014). In this study, garlic leaves showed efficacy at 5%
juice (81.8%), followed by 5% dust (64.2%), 10% juice
(63.1%) and 10% dust (50.0%) against A. galli. These records have close agreements with study of Murdiati et al.,
(1997), Lamtiur (2000) and Rahman (2002) that proved
the anthelmintics efficacy against A. galli. These findings
have also similarities with the study of Kumar et al (1991)
who compared in vitro effects of BITC (benrylisothiocyanate), an anthelmintic principle of garlic with mebendazole against A. galli and found effective.
The leaves of C. gigantea (Akanda) are used as a vermicide. The active component of C. gigantea is calotropain.
This proteolytic enzyme can digest parasites (Lechat et al.,
1978). The efficacy of leaves of akanda is determined in
this experiment. The leaves of akanda revealed the maximum efficacy against eggs of A. galli at 5% dust (75.0%)
followed by 10% juice (73.0% and 69.2%) both at 10%
juice and 20% dust concentration.. These records is more
or less similar with the findings of Lal et al. (1976) and
Ali (2006) who studied the anthelmintic efficacy of akanda
against adult A. galli as well as other helminthes. Similarly,
Rahman (2002) recorded the efficacy of akanda in aqueous
(67%) and ethanol extract (79%) against gastro intestinal
nematodes in vivo. These findings are partially agreed with
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The Journal of Advances in Parasitology
percentage of efficacy of present study, though present
study recommends the plants as effective which have at
least 70% efficacy.
Preliminary phytochemical investigation of methanolic extract revealed the presence of alkaloid, acetogenin,
flavonoids, stenoid and triterpenoids in A. reticulata (Krishnadev et al., 2010). Previous studies reported that alkaloid is responsible for the paralysis of earthworm. Tannin
might have anthelmintic activity by binding with free proteins in gastrointestinal tract of earthworm and cause death
(Tiwari et al., 2011; Rubini et al., 2012). Dust powder of
custard apple was found 57.1% and 53.3% effective at 5%
and 10% concentration respectively. These results could be
compared with the findings of Chakraborty et al. (2008)
who recorded the efficacy (77.0%) of custard apple leaves
against sporulation of Eimeria tenella oocysts. According to
Bhale et al. (2011), methanolic extract of leaves of custard
apple have a potent anthelmintic activity.
CONCLUSION
It is concluded that among five plants and different preparations, pineapple, turmeric and akanda were found effective. But they can not be used directly, as many microbes
multiply in moist liter. So further studies are therefore
needed to find out the side effects of these plants and determine the recommended doses in poultry.
ACKNOWLEDGEMENTS
Authors express special thanks to respected teachers in the
department of Parasitology for their kind help.
CONFLICT OF INTEREST
The authors state no conflict of interest.
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